In situ localization of mitochondrial DNA replication in intact mammalian cells

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.     

Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA

The organisation of mammalian mitochondrial DNA (mtDNA) is more complex than usually assumed. Despite often being depicted as a simple circle, the topology of mtDNA can vary from supercoiled monomeric circles over catenanes and oligomers to complex multimeric networks. Replication of mtDNA is also not clear cut. Two different mechanisms of replication have been found in cultured cells and in most tissues: a strand-asynchronous mode involving temporary RNA coverage of one strand, and a strand-coupled mode rather resembling conventional nuclear DNA replication. In addition, a recombination-initiated replication mechanism is likely to be associated with the multimeric mtDNA networks found in human heart. Although an insight into the general principles and key factors of mtDNA organisation and maintenance has been gained over the last few years, there are many open questions regarding replication initiation, termination and physiological factors determining mtDNA organisation and replication mode. However, common themes in mtDNA maintenance across eukaryotic kingdoms can provide valuable lessons for future work.     

Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity

The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNA-expressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous single-strand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to γ-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observaion, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.     

Comparison of plastid DNA replication in different cells and tissues of the rice plant

In a previous study, we mapped replication origin regions of the plastid DNA around the 3 end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.     

Prominent mitochondrial DNA recombination intermediates in human heart muscle

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.     

Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space.

Mammalian mitochondria contain strong nuclease activity. Endonuclease G (endoG), which predominantly resides in mitochondria, accounts for a large part of this nuclease activity. It has been proposed to act as an RNase H-like nuclease on RNA.DNA hybrids (R-loops) in the D-loop region where the origins of mitochondrial replication are mapped, providing RNA primers for mtDNA replication. However, in contrast with this proposed activity, endoG has recently been shown to translocate to nuclei on apoptotic stimulation and act as a nuclease without sequence specificity. To clarify the role of endoG in mtDNA replication, we examined its submitochondrial localization and its ability to cleave R-loops. At low concentration, it preferentially produces double-stranded breaks in R-loops, but does not act as an RNase H-like nuclease. In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs. These results do not support the involvement of endoG in mtDNA replication. Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double-stranded breaks in R-loops, we propose that three-stranded DNA may be the preferred substrate of endoG.     

Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells

Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively dividing cells. By electron microscopy we observed rearranged mtDNA molecules in cultured human cells expressing a catalytically impaired helicase. As these molecules were undetectable by PCR, we propose that deleted mtDNA molecules in cultured cells are fragile and sensitive to heating. Further consequences of mtDNA replication stalling are discussed.     

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

Normal reconstruction of DNA supercoiling and chromatin structure in cockayne syndrome cells during repair of damage from ultraviolet light.

The chromatin of human cells undergoes structural rearrangements during excision repair of ultraviolet damage in DNA that were detected by transient relaxation of DNA supercoiling and increased staphylococcal nuclease digestibility of repaired sites. Inhibition of polymerization and/or ligation of repaired regions with inhibitors of DNA polymerase alpha (cytosine arabinoside and aphidicolin) resulted in the accumulation of single-strand breaks, delayed reconstruction of DNA supercoiling, and maintenance of the staphylococcal nuclease digestibility. These observations suggest that reconstruction of the native chromatin state requires completion of repaired regions with covalent ligation into the DNA strands. Although previous claims have been made that a late stage associated with ligation of repaired regions may be defective in cells from patients with Cockayne syndrome, complete reconstruction of the native chromatin occurred in cells from three unrelated patients after ultraviolet irradiation. No abnormality in repair was therefore detected in Cockayne syndrome cells. The hypersensitivity of cell survival and semiconservative DNA replication to damage by ultraviolet light in this human disorder must therefore be regarded as features of a primary defect in DNA metabolism unrelated to DNA repair.     

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli.

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.     

The human mitochondrial replication fork in health and disease

Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.     

Excision of uracil from bromodeoxyuridine-substituted and U.V.-irradiated DNA in cultured mouse lymphoma cells.

A uracil-DNA glycosylase activity was detected in cell-free extracts from cultured mouse lymphoma L5178 cells. We investigated whether or not this enzyme plays a role in the removal of uracil from chromosomal DNA. U.V. light (254nm) irradiation of the cells with BUdR-substituted DNA produced not only single-strand breaks but also 'internal' uracil residues that were recognized as substrate sites by uracil-DNA glycosylase. These 'internal' uracil residues were lost from the DNA upon reincubation of the irradiated cells. The product released from the DNA was identified as uracil. Thus, the intracellular action of the uracil-DNA glycosylase was demonstrated and the subsequent reconstitution of the DNA strand was inferred in cultured mammalian cells.     

Mitochondrial dysfunction in cancer cells due to aberrant mitochondrial replication

Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.